A gene is considered a candidate gene for obesity or type 2 diabetes (T2D) in Pima Indians if 1) it has a known physiological function in a pathway relevant to T2D/obesity or 2) it is associated with diabetes/obesity in another human population or in an animal model. In the past year we have directly sequenced and genotyped all tagging variants in several physiologic candidate genes for association analysis with BMI or T2D. Genotyping was performed in two large population-based samples of individuals collected from the Gila River Indian Community. Rare variants in genes along the Leptin/Melanocortin pathway cause severe monogenic obesity in childhood; therfore, genes along this pathway have been sequenced and genotyped as candidate genes for obesity in Pima Indians. Within the past year, we have studied the genes MC4R,Sim1 and BDNF. We have previously reported on rare coding variation in MC4R, but have now investigated whether common, non-coding variation at this locus also affects risk for obesity. A common haplotype (frequency=0.35) from two independent signals in MC4R was identified which provided evidence for association with BMI in American Indians during both adulthood and childhood. Our longitudinal data with repeated BMI measures allowed a comprehensive assessment for the effect of MC4R on obesity risk over lifetime (age 5-50 years). We also identified a MC4R promoter variant that affects expression in vitro, and showed that it influences risk of obesity in part through a propensity for increased food intake and decreased energy expenditure. Our study was the first to report association between a common variant and ad libitum food intake using an automated vending machine paradigm in a clinical research unit, which provides a more objective, reproducible and accurate measure of food intake than that afforded by methods based on self-report. We also analyzed the gene BDNF and perfomed a meta-analysis of our data with that of other another group studying obesity in Caucasian children. Glucokinase (GCK) plays a role in glucose metabolism and glucose-stimulated insulin secretion. Rare mutations in GCK cause maturity-onset diabetes of the young (MODY). We investigated whether common variation (mAF 0.01) in GCK is associated with metabolic traits and T2D and identified a novel 3UTR SNP chr7:44184184-G/A that associated with the rate of carbohydrate oxidation postabsorptively (=0.22 mg kgEMBS-1 min-1, p=0.005) and during a hyperinsulinemic, euglycemic clamp (=0.24 mg kgEMBS-1 min-1, p=0.0002), the rate of carbohydrate oxidation in a respiratory chamber (=311 kJ/day, p=0.03) and 24-h energy expenditure that was attributable to the thermic effect of food (=520 kJ/day, p=3.39x10-6). This 3UTR SNP was also associated with diabetes OR=1.36(1.11, 1.65), p=0.002, where the A-allele (AF=0.05) was associated with a lower rate of carbohydrate oxidation, lower 24-h energy expenditure and higher risk for diabetes. In a Cox proportional hazards model, a lower than the mean rate of insulin-stimulated carbohydrate oxidation at baseline predicted a higher risk for developing diabetes than those above the mean HRR=2.2(1.3-3.6), p=0.002. Genetic variants in SLC16A11 were recently reported to be associated with T2D in Mexican and other Latin American populations. The diabetes risk haplotype had a frequency of 50% in Native Americans from Mexico, but was rare in Europeans and Africans. We analyzed SLC16A11 in 12,811 North American Indians and found that the diabetes risk haplotype, tagged by the rs75493593 A allele, was nominally associated with T2D; however, there was a strong interaction with BMI such that the diabetes association was stronger in leaner individuals. Rs75493593 was also strongly associated with BMI in individuals with T2D (P = 3.4 10-15) but not in non-diabetic individuals (P = 0.77). Longitudinal analyses suggest that this is due, in part, to an association of theA allele with greater weight loss following diabetes onset. Analyses of global gene expression data from adipose, skeletal muscle, and whole blood provide evidence that rs75493593 is associated with expression of the nearby RNASEK gene, suggesting that RNASEK expression may mediate the effect of genotype on diabetes. Missense variants in KCNJ11 and ABCC8, which encode the KIR6.2 and SUR1 subunits of the -cell KATP channel, have previously been implicated in type 2 diabetes, neonatal diabetes, and hyperinsulinemic hypoglycemia of infancy (HHI). To determine whether variation in these genes affect risk for type 2 diabetes, or increased birth weight as a consequence of fetal hyperinsulinemia in Pima Indians, missense and common non-coding variants were analyzed in individuals living in the Gila River Indian Community. A R1420H variant in SUR1 (ABCC8) was identified in 3.3% of the population (N = 7,710). R1420H carriers had higher mean birth weights, and a twofold increased risk for type 2 diabetes with a 7 year earlier onset age despite being leaner than non-carriers. One individual homozygous for R1420H was identified; retrospective review of his medical records was consistent with HHI and a diagnosis of diabetes at age 3.5 years. In vitro studies show that the R1420H substitution decreases KATP channel activity. Identification of this loss-of-function variant in ABCC8 with a carrier frequency of 3.3% impacts clinical care since homozygous inheritance and potential HHI will occur in 1/3,600 births in this American Indian population. The PTF1 complex is critical for pancreatic development and maintenance of adult exocrine pancreas. Therefore, we analyzed variation in genes that form this complex, namely PTF1A, RBPJ, and its paralogue RBPJL. A Thr280Met variant (rs200998587, risk allele frequency = 0.03) in RBPJL, identified only in Amerindian-derived populations, had a nominal association with T2D (OR = 1.601.21-2.13 per Met allele, P = 0.001) and age of diabetes onset (HR = 1.401.14-1.72, P = 0.001). Knockdown of Rbpjl in mouse acinar cells resulted in a significant decrease in the expression of Cel and Ctrb. Interestingly, CTRB1/2 is a previously reported T2D locus where the protective allele associates with increased GLP-1 stimulated insulin secretion and higher expression of CTRB1/2. Further in-vitro studies showed that cells expressing the Met280 allele had lower RBPJL protein levels than cells expressing the Thr280 allele, despite having comparable levels of RNA, suggesting that the Met280 protein is less stable. Luciferase assays in HEK293 cells which examined two different RBPJL responsive promoters, including the promoter for CTRB1, identified reduced transactivation by the Met280 RBPJL. Similarly, overexpression of both Met280 and Thr280 RBPJL in mouse acinar cell identified a significant impairment in the expression of Cel when transactivated by the Met280 RBPJL. Therefore, we believe that this functional Thr280Met variant in RBPJL modifies T2D risk by regulating exocrine enzyme expression. We have also identified a novel missense variant (G310D) in the candidate gene IGF1R; this variant occurred in 6% of Pima Indians but did not occur in 2,687 American Indians who also live in Arizona but are from non-Pima tribes. Therefore, we believe this variant, which is not reported in any public databases, may be private to Pima Indians of Arizona. Genotyping of the G310D in a population-based sample of Pima Indians showed that it associates with a 2.3-fold increased risk for T2D, and an earlier age of T2D onset in women, but not men. Consistent with the previously defined role of IGF1R in prenatal and postnatal growth, subjects carrying the T2D risk allele D at G310D also had lower birth weights and shorter stature in adulthood as compared to subjects homozygous for the non-risk allele.